Summary: The speed, resolution, and mass accuracy of modern mass spectrometers have revolutionized proteomics, but the accurate identification and quantification of post-translational modifications (PTMs) remain a major challenge that ultimately limits many current biomedical and pharmaceutical applications. A pivotal weakness lies in the almost exclusive use of collision-induced dissociation (CID) to induce fragmentation because most PTMs, such as phosphorylation, have labile bonds that are commonly lost in complex ways when subjected to CID. Furthermore, CID limits proteomics to bottom-up analyses of trypsin- digested peptides of 10-40 residues. It is well established that an alternative fragmentation methodology called electron capture dissociation (ECD) can produce exceptionally clean spectra that preserve PTMs, but this technique is currently feasible only in expensive FTICR mass spectrometers. Providing enough low- energy electrons to efficiently fragment peptides has, until now, fundamentally limited the application of ECD. We have developed an ECD cell that operates without affecting the ion-flight path of conventional mass spectrometers. Based on that new technology, our Phase I SBIR project was designed to at least double fragmentation efficiency by exploiting the distinctive geometry of Orbitrap mass spectrometers to enable ions to make two passes through the ECD cell. We exceeded our Phase I milestones by demonstrating that our ECD cell quadrupled efficiency, due in part to ions moving slower through our cell in the Orbitrap than in other types of mass spectrometers. We further showed that our ECD cell was easily installed in Orbitraps in an hour without affecting the instruments' performance. We established the ECD works particularly well for the analysis of native proteins, even for top-down hydrogen/deuterium structural analyses. For Phase II, our 1st aim is to refine each of the elements in the ECD cell to integrate easily in four Orbitrap family members and then to exploit the cell's capabilities to produce high-energy electrons to achieve stronger fragmentation by Electron-Induced Dissociation (EID). Our 2nd aim involves working with early-adopters to develop the technology for commercial release and validate its substantial advantages over competing technologies. Adoption of our technology will accelerate the ability of many NIH investigators to probe disease mechanisms and identify diagnostic/therapeutic biomarkers with increased speed and accuracy that will result in fewer mistaken identifications in complex biological samples. Our immediate commercial objective for Phase-III is to provide cost-effective upgrade kits for the 6,000 Orbitraps in service. The longer- range commercial goal is to develop fully integrated solutions that will enable the biopharmaceutical industry to characterize therapeutic protein products such as antibody-conjugated drugs, and to validate ?biosimilars? for the FDA and other regulatory agencies.